With this research we set out to develop a coumarin-based novel fluorescent probe NTR-AHC for the detection of biological thiols and nitroreductase (NTR). Probe NTR-AHC was constructed by attaching the NTR trigger moiety (p-nitrobenzyl) and biothiol acceptor (maleic acid monoamide), to the core fluorophore AHC. In the presence of both glutathione (GSH) and NTR, probe NTR-AHC produced a >8-fold increase in fluorescence intensity at 463 nm.